NAME: _______________ 7.06 Cell Biology EXAM #4 May 20, 2004 1 of 14 7.06 Cell Biology EXAM #4 May 20, 2004 This is an open book exam, and you are allowed access to books and notes. Please write your answers in pen to the questions in the space allotted; if you use the back of a sheetmake it clear which answer is where. And be sure to put your name on each page in casethey become separated! Good luck! Name: Student ID #: Question 1. ____________ Question 2. ____________ Question 3. ____________ Question 4. ____________ Question 5. ____________

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NAME: _______________ 7.06 Cell Biology EXAM #4 May 20, 2004 2 of 14 1. (15 points) Your friend, who works at a research station in the arctic sea has discovered a new organism, and has raised antibodies against random proteins of this organism. When he examined the localization pattern of these proteins within cells he discovers that two proteins reveal an identical filament-like staining pattern within the cells. He sends you the pictures and you tell him that the filaments remind you of microtubules. In his reply e-mail he asks you whether you can suggest an experiment to test this hypothesis. a. ( 3 pts ) Briefly, suggest an experiment to test whether the two proteins your friend identified form heterodimers, employing the antibodies he raised against the proteins. He could do a co-immunoprecipitation experiment. Immunoprecipitate one protein and test whether the other protein co-immunoprecipitates. Having established that the proteins indeed form heterodimers and having discovered that the proteins bind GTP you decide to call the proteins alt – α protein and alt-β protein (alt for almost –like – tubulin). Presently, you would like to investigate the polymerization kinetics of alt filaments. To this end you perform an elongation assay with non-fluorescent seed and fluorescently labeled alt heterodimers. Here are the results you obtain after the reaction has reached steady state : b. ( 2 pts ) What does this experiment tell you about the critical concentration for assembly for the left and right end of the filament? The critical concentration is the same for both ends. 0.1 mm 0.1 mm seed

NAME: _______________ 7.06 Cell Biology EXAM #4 May 20, 2004 3 of 14 In the mean time your friend was busy purifying alt-associated factors. He succeeded in purifying three of them, factor X, factor Y and factor Z. He asks you to add these proteins to your in vitro filament formation assay. Here are the results you obtain after the reaction has reached steady state: c. ( 6 pts ) How do these factors affect alt filament assembly? Factor X: decreases the critical concentration of the right end Factor Y: Appears to function as a left end capping factor. Factor Z:

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